Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells (Treg). We have demonstrated that the regulatory T cells can be easily identified in normal lymphoid tissues by expression of CD4, the interleukin-2 receptor alpha chain (CD25), and the transcription factor, FoxP3. Transfer of CD4+ CD25- Foxp3- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+Foxp3+ T cells. The major goals of this project are to define the function and mechanism of action of Treg cells in vivo: (1) We have analyzed the effects of polyclonal Treg on the development of autoimmune gastritis (AIG). Treg inhibited the development of disease, but failed to inhibit the migration of the effector cells into the gastric lymph node (gLN) or into the stomach. Notably, Treg did not inhibit the expansion of autoreactive T cells in the gLN. The primary effect of the Treg was inhibition of the differentiation of the autoantigen-specific T cells to Th1 effector cells as reflected by a decrease in antigen-stimulated IFN-gamma production and reduction in Tbet expression. (2) We have previously demonstrated that the target antigen in AIG is the H+/K+ATPase, the proton pump of the gastric parietal cell. Studies over the past several years have demonstrated that stimulation of naive polyclonal T cells in the presence of TGF-beta results in the induction of Foxp3 expression and T regulatory activity in vitro and in vivo. We have demonstrated that naive H+/K+ ATPase-specific CD4+CD8-Foxp3- thymocytes from TCR transgenic mice can be easily expanded and converted to FoxP3+ T cells when activated in the presence of TGF-beta. TGF-beta-induced Tregs (iTregs) resemble thymic?derived Treg as they are anergic, suppressive, and have a reduced capacity to produce effector cytokines upon restimulation in vitro. Most importantly, these autoantigen-specific iTregs were able to potently inhibit the development of AIG. iTregs prevented the priming and expansion of the effector T cells very early after cell transfer and appeared to target DC presenting endogenous autoantigen. In autoimmune disease, it is likely that the frequency of autoantigen-specific CD4+Foxp3- T cells would be greater than autoantigen-specific CD4+Foxp3+ nTreg. Taken together, these data demonstrate that it is possible to convert potential autoantigen-specific effector T cells to potent autoantigen-specific iTregs. A similar approach might be applicable to the treatment of autoimmune disease in man. 3) Although CD4+CD25+ T regulatory cells (Treg) play an essential role attenuating immune responses, little is known regarding the normal behavior of Treg in the context of an inflammatory response. Approximately 3% of Foxp3+ Treg incorporate BrdU following a single pulse in vivo. The dividing cell population is moderately (2-3 fold) enriched in the subset (20-30%) of Treg that co-express Foxp3 and CD103 and greatly (4-6 fold) enriched in the subset of CD103+ Treg that express the NK antigen, KLRG1. These subsets are enriched to the same extent in the dividing cell population seen after transfer of Treg to normal recipients, or in the draining lymph node following injection of Toll receptor ligands. CD103+, but not CD103-, Foxp3+ Treg cells are capable of mediating antigen non-specific suppression of T cell activation directly ex vivo in the absence of TCR engagement. CD103+Foxp3+ Treg are present in normal numbers in MHC class II -/- and MHC class I -/- animals, but greatly reduced in MHC class I/II -/- mice. Surprisingly, the CD103+KLRG1+ subset is maintained at normal levels in MHC class I/II -/- mice. Collectively, these studies indicate that a subset of Treg may be activated in vivo by non-MHC encoded molecule(s), the expression of which is regulated by inflammatory stimuli. Thus, ?innate? suppressor T cells capable of recognizing ligands distinct from those driving the effector T cell response may play an essential role suppressing inflammation. (4) In collaboration with Drs. Moudgil and Bala, we have studied the role of antigen specificity in the generation as well as the suppressive function of Treg cells. Wild type (WT), but not mouse lysozyme-M (ML-M) knockout (MLKO), mice are tolerant to a challenge with ML-M. WT mice depleted of CD4+CD25+ T cells gain responsiveness to ML-M, and the major epitopes of ML-M revealed are the same as those responded to by unmanipulated MLKO mice immunized with ML-M. Furthermore, MLKO mice depleted of CD4+CD25+ T cells develop T cell response to two new epitopes of ML-M that are not at all revealed either in unmanipulated WT or in CD4+CD25+ T cell-depleted WT mice. Thus, mice lacking ML-M were not deficient in the generation of CD4+CD25+ T cells that could efficiently suppress ML-M-primed target T cells. However, WT and MLKO mice displayed differences in both the epitope specificity and the relative avidity for antigen recognition of the target T cells regulated by CD4+CD25+ T cells. These differences, superimposed on the deletion of certain ML-M-specific T cell subsets following thymic tolerance, imprint the unique profiles of T cell response against self lysozyme in WT and MLKO mice. These results are of significance in further understanding of the role of CD4+CD25+ T cells in the induction of tolerance as well as the regulation of autoreactivity of self epitope-specific T cells.